Our lab is dealing with some very pesky contamination in our negative (water only) controls lately. It seems in some of our assays we are seeing the wildtype band, the transgenic band, or even both. The most confusing part is the inconsistency of it all. We can run three assays together, each with different primer sets and only one will show the contamination. That means the only thing changing is the primers, but we will have used the same primers in the past with no problems. Unless we have all suddenly forgotten how to do PCR, there is something fishy going on. Notes:

dNTPs have worked fine for other Assays and QPCR

We cleaned our pipets and always use barrier tips

Can anyone offer any advice? If we could at least pinpoint where it is coming from, that would help. Thanks!

The samples (and/or other solutions) are contaminated with wildtype DNA?

One must presume that long and short arguments contribute to the same end. - Epicurus...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

Right now we are to the point where when it deosn't work we just have to re-do it and hope for the best. Everytime it doesn't work we just replace the primers just in case, but I just don't think it's that. I honestly am starting to think it's our water aliquots. We just got these new colorful 1.5 ml tubes and they don't really close all the way. I think that is a link and I'm just going to blame the ungergrads for now too I think.

Sounds like you are isolating tail clips to determine whether your transgene is present? Did someone double dip in the phenol or other solution for DNA preparation. If you use NaOAc for precipitation, it is possible that you have contaminants floating bound to the salt that only get picked up a portion of the time. I don't know. Sounds fishy either way.

How close is the PCR preparation area and the gel area? If the room is not well ventilated and you ran the gel close to the PCR preparation - you may have some of the PCR fragments just flying around.

We had similar problem in our lab, and now we prepare PCR/qPCR in separate room, and runing gels in other lab (even changing lab coats) in 2 years we had only few cases of negative control giving a band.